This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. HIV particles bud from host cells by recruiting machinery of the cellular MVB pathway, which normally serves to bud vesicles into late endosomeal compartments. We propose to study three cellular protein components of the MVB pathway that function in HIV budding: (1) Vps4, (2) ALIX, and (3) ESCRT I. In all cases some crystal structures are available, and SAXS is expected to further understanding of functionally important assembly states and conformational changes. Vps4 is an AAA-ATPase and is the only enzyme of the MVB pathway. We will use SAXS to build a model for the functionally relevant oligomerization state, determine the disposition of the substrate-binding N-terminal domains, and determine the structural basis for its interaction with its functionally relevant binding partner Vta1. SAXS analysis of ALIX should reveal how the N-and V domains of this scaffolding protein are organized and test the proposal that the two arms of the V-domain structure move in response to ligand binding. For human ESCRT-I, we aim to determine the overall organization of component domains/subunits and test the proposal that ligand binding induces deautorepression via a conformational change. Our preliminary data include crystal structures of human VPS4B and yeast Vps4 in monomeric conformations and domains of ALIX and ESCRT I. Proteins and complexes have been characterized by sizing chromatography and analytical ultracentrifugation. Rapid access SAXS data have demonstrated the feasibility of studies on Vps4.